Interleukin-2 (IL-2), a lymphokine which is produced by normal peripheral blood lymphocytes and induces proliferation of antigen or mitogen stimulated T cells after exposure to plant lectins, antigens, or other stimuli, was first described by Morgan, D. A., et al., Science (1976) 193:1007-1008. Then called T cell growth factor because of its ability to induce proliferation of stimulated T lymphocytes, IL-2 modulates a variety of functions of immune system cells in vitro and in vivo. IL-2 is one of several lymphocyte-produced messenger-regulatory molecules that mediate immunocyte interactions and functions.
IL-2 was initially made by cultivating human peripheral blood lymphocytes (PBL) or other IL-2-producing cell lines. See, for instance, U.S. Pat. No. 4,401,756. Recombinant DNA technology has provided an alternative to PBLs and cell lines for producing IL-2. Taniguchi, T., et al., Nature (1983) 302:305-310 and Devos, R., Nucleic Acids Research (1983) 11:4307-4323 have reported cloning the human IL-2 gene and expressing it in microorganisms.
Native human IL-2 is an antigen-nonspecific, genetically unrestricted soluble factor produced by erythrocyte rosette positive T cells stimulated with antigens, mitogens and alloantigens. It is a protein with a reported molecular weight in the approximate range of 13,000 to 17,000 daltons (S. Gillis and J. Watson, J Exp Med (1980) 159:1709) isoelectric point in the approximate range of pH 6-8.5. Human IL-2 has a number of in vitro and in vivo effects including enhancing the proliferative responses of human peripheral blood mononuclear cells or murine thymocytes, enhancing the immune response in humans and in animals against bacterial, parasitic, fungal, protozoan and viral infections, and supporting the growth of continuous T cell lines.
Human IL-2 has been obtained from genetically engineered E. coli as an unglycosylated protein with biological activities equivalent to those of native, glycosylated IL-2. [Taniguchi et al., Nature, 302 (5906): 305-310 (Mar. 24, 1983)., Devos et al., Nuc. Acids Res., 11:4307-4323 (1983); Rosenberg et al., Science, 223:1412-1415 (1984); Wang et al., Science, 224:1431-1433 (1984); and Doyle et al. J. Biol. Resp. Modifiers, 4:96-109 (1985)]. Rosenberg and his co-workers have shown that systemic administration of recombinant IL-2 in high doses causes regression of established metastases in mice [Rosenberg et al., J. Exp. Med., 161:1169-1188 (1985)]; and, in conjunction with lymphokine-activated killer cells, in humans [Rosenberg et al., New Eng. J. Med., 313:1485-1492 (1985)].
U.S. Pat. No. 4,518,584 discloses muteins (analogs) of IL-2 in which the cysteine normally occurring at position 125 of the wild-type or native molecule has been replaced with a neutral amino acid, such as serine or alanine. Such muteins possess the biological activity of native IL-2. The patent discloses that for therapeutic or diagnostic applications, such IL-2 muteins may be formulated in nontoxic, nonallergenic, physiologically compatible carrier media such as distilled water, Ringer's solution, Hank's solution, physiological saline, and the like. Administration of the IL-2 analogs to humans or animals may be oral or intraperitoneal or intramuscular or subcutaneous as deemed appropriate by a physician. The amount of IL-2 mutein administered will usually range between about 1.times.10.sup.4 and 2.times.10.sup.8 units. EP 200,280 (published Oct.12, 1986) discloses muteins of IL-2 whereby the methionine at position 104 has been replaced by a conservative amino acid.
Microbially produced IL-2 is not glycosylated and is produced in a reduced state by microorganisms. When purified and oxidized (cystine form) the microbially produced IL-2s exhibit activity comparable to native human IL-2.
Procedures for purifying native IL-2 from T cells are described by Watson et al., J Exp Med (1979) 150:849-861; Gillis et al., J Immunology (1980) 124:1954-1962; Mochizuki et al., J Immun Meth (1980) 39:185-201; Welte et al., J Exp Med (1982) 156:454-464; and European published patent applications 92163 and 94317. In general, these procedures involve precipitating proteins from culture supernatants with ammonium sulfate followed by a chromatographic fractionation.
Commonly owned U.S. Pat. No. 4,569,790 to K. Koths et al. describes a process for recovering IL-2 from an IL-2 producing microorganism whereby the microorganism cell membrane is disrupted, the disruptate is extracted with an aqueous solution of a chaotropic agent such as urea, the IL-2 is solubilized with a surfactant, e.g., sodium dodecyl sulfate (SDSl, and the IL-2 is separated in the presence of a reducing agent.
Purification and activity assurance of precipitated heterologous proteins is also described by U.S. Pat. Nos. 4,511,502; 4,511,503; 4,512,922; 4,518,526 and 4,599,127; and EP 114,506.
Commonly owned U.S. Pat. No. 4,604,377 to Fernandes et al. discloses a formulation of a purified microbially produced recombinant IL-2 in which the IL-2 is admixed with a water soluble carrier, such as mannitol, and a sufficient amount of a surface active agent such as SDS or sodium deoxycholate to ensure solubility of the recombinant IL-2 in water at physiological pH.
Many heterologous proteins are precipitated intracellularly in the form of refractile or inclusion bodies which appear as bright spots visible within the enclosure of the cell under a phase contrast microscope at magnifications down to 1000 fold. See e.g., Miller et al., Science (1982) 215:687-690; Cheng, Biochem. Biophys. Res. Comm., (1983) 111:104-111; Becker et al., Biotech. Advs. (1983) 1:247-261; Kleid et al., ch. 25 in Developments in Industrial Microbiology, Vol. 25, p. 317-325 (Society for Industrial Microbiology, Arlington, VA, 1984) and Marston et al., Bio/Technology (September, 1984), pp. 800-804.
EP 206,828 (published Dec. 30, 1986) discloses improved methods for recovering and purifying refractile bodies containing recombinant protein.
Wang et al., J. Parenteral. Drug Assoc., 34, 452-462 (1980) provides a review of excipients and pHs for parenteral products used in the United States. A list of solubilizing agents such as detergents and lipids in use for various drugs is provided in Table I thereof and under section II entitled "Solubilizers, Wetting Agents or Emulsifiers" of that table, polyethylene glycol 300, polysorbate 20, 40 and 80, and propylene glycol among others are listed for a variety of pharmaceuticals.
U.S. Pat. No. 4,645,830 discloses IL-2 compositions comprising a solution with human serum albumin, and a reducing compound adjusted to a pH range of 3 to 6.
Japanese Kokai Application 61/293926 (published Dec. 24, 1986) discloses interleukin-2 compositions containing surfactants (Tween.RTM. 20, Tween.RTM. 80, HCO 60, Brij 35, Triton X-100) as stabilizers.
Morikawa et al., Cancer Res., 47:37-41 (Jan. 1, 1987), reports on the use of Pluronic F-127 gel (a polyoxyethylenepolyoxypropylene surface active block copolymer) as a sustained release vehicle for topical administration of recombinant interleukin-2.
Copending, commonly owned, U.S. application Ser. No. 775,751, filed Sept. 13, 1985 entitled "An Improved Formulation for Lipophilic Proteins" outlines a high pH and a low pH process for recovering and purifying lipophilic recombinant proteins such as HIFN-and .beta. and interleukin-2 from host strains to yield a protein preparation which may be formulated into a stable pharmaceutical composition. Said composition carrying a therapeutically effective amount of the biologically active lipophilic protein dissolved in a non-toxic, inert, therapeutically compatible aqueous-based carrier medium at a pH of 6.8 to 7.8 also contains a stabilizer for the protein, such as human serum albumin, human serum albumin and dextrose, and human plasma protein fraction.
EP 217,645 (published Apr. 8, 1987) discloses pharmaceutical compositions containing IFN-.beta. or interleukin-2 dissolved in a stable carrier medium at pH 7.0 to 8.0 stabilized with sodium laurate.
WO 87/00056 (published Feb. 15, 1987) discloses pharmaceutical compositions wherein recombinant IFN-.beta., IL-2 or an immunotoxin is dissolved in an aqueous carrier medium without the presence of a solubilizing agent. Such unconjugated proteins are insoluble in water at pH 6-8 without a solubilizing agent. The protein is solubilized by selectively conjugating it via a coupling agent to a water-soluble polymer selected from polyethylene glycol homopolymers or polyoxyethylated polyols.
There remains a need in the art for formulations of biologically active, recombinant interleukin-2 that are pure enough for clinical administration but substantially or totally free of residual strong detergents or chaotropes, such as SDS, used in the extraction and purification processes. Further, there is a need for formulations that provide alternatives to those containing non-IL-2 protein, such as those containing albumin.
Further, alternative processes for preparing formulations containing biologically active, recombinant interleukin-2 which avoid very high pH ranges are desirable. The instant invention meets such needs.